Alport syndrome results from mutations in either the type IV collagen COL4A3, COL4A4, or COL4A5 genes. Pathology includes a juvenile onset progressive glomerulonephritis with glomerular basement membrane (GBM) rarefication and expansions of the mesangial matrix, culminating in death due to renal failure. A gene-knockout mouse model for Alport syndrome was produced in this laboratory, the renal pathology of which is remarkably similar to that observed in humans. Deposition of specific extracellular matrix proteins in the GBM follows course with basement membrane rarefication. This must be due to changes in their synthesis and/or turnover.. The aims of this proposal focus on clarifying the role of synthesis and degradation of basement membrane collagens and associated proteins in Aport GBM disease progression. The possible role of integrin receptors, transforming growth factor beta 1 (TGF-beta1), and metalloproteinases (and their inhibitors) will be explored. Quantitative analysis (employing material from isolated glomeruli) will be used to determine relative levels of specific mRNAs (by norther blot) and their corresponding proteins (by western blot). In situ hybridization studies will determine which glomerular cell types synthesize these proteins. Observations with potential mechanistic significance will be tested using human renal biopsy tissues from normal and Alport patients. Whether TGF-beta1 or proteinuria is linked to induction of genes encoding extracellular matrix molecules will be probed using a combination approach of TGF-beta type I and II receptor inhibitor (FK506) molecules will be probed using a combination approach of TGF-beta1. A double knockout of the COL4A3 and integrin alpha1 chain has been produced, and shows a marked reduction in the rate of Alport renal disease progression. The molecular mechanism of this surprising effect will be explored. Identification of the molecular mechanisms underlying the imbalance in GBM homeostasis in Alport syndrome may reveal potential targets for pharmacologic intervention.